Gel electrophoresis is a widely used method for separating biomolecules, such as proteins, peptides, nucleic acids etc. Gel electrophoresis involves the migration of electrically charged molecules in an electric field. A solution containing biomolecules is placed in contact with a supporting gel, an electric field is applied and the molecules are allowed to migrate on or through the electrophoretic gel. Electrophoretic separation of molecules is based on the difference in charge density of the molecules as well as the sieving effect of the porous gel media. The extent of sieving depends on how well the pore size of the gel matches the size of the migrating molecules.
Separation gels have often been prepared by polymerising monomers comprising one or more polymerisable unsaturated structures (primarily carbon—carbon double bonds), in particular vinyl groups. When monomers having two or more polymerisable unsaturated structures are present in the polymerisation mixture, cross-linked polymers will form. Typically the monomers have been acrylates, methacrylates, acrylamides, methacrylamides, acrylonitrile, methacrylonitrile, if applicable bisforms thereof, etc. In certain cases the monomer has been a polymer (prepolymer) carrying a plurality of unsaturated structures. Typical prepolymers have been selected among polyhydroxypolymers, such as dextran, agarose and other polysaccharides. See U.S. Pat. No. 4,094,832; U.S. Pat. No. 4,094,833; EP 87995; WO 9731026, WO 9726071.
Previous polymerisation systems utilise various kinds of initiators. Typical initiators are chemical and thermal. Thermal initiators are often preferred. They have their best efficiency in the range of 50–90° C. Well-known chemical/thermal initiators are azo compounds (for instance 2,2′-azobis(2,4-dimethylvaleronitrile), azoisonitriles, peroxides (for instance benzoylperoxide), persulphates. One important kind of chemical initiators requires irradiation, for instance V, in order to start a polymerisation. Redox systems have also been used, for instance Fenton's reagent (hydrogen peroxide+Fe2+).
There have been problems associated with the production of separation gels. Acryl amides, for instance, are often toxic. This is of particular importance for electrophoretic gels because the customer himself often cast them. Further acrylates and acrylamides are sensitive to hydrolysis. Still further, electrophoretic gels have specific demands on homogeneity and physical stability that sometimes can be problematic to achieve. The amount of residual monomer in the polymer immediately after polymerisation has often been high requiring subsequent washing steps and the like.
Consequently, there is a need for new separation gels, for instance electrophoretic gels, and/or improved methods for the production thereof.
Akashi M et al. (Kuromatogurafi 15(2) (1994) 108–109) have tested a gel obtained by radiation initiated polymerisation of N-vinyl acetamide and a bisform of an acrylate in capillary electrophoresis (CE).
Akashi M et al. (JP application 96-43050 (1996) have tested a separation medium obtained by thermally initiated polymerisation of vinyl amine or N-vinyl acetamide (azobis initiator). Koshiji J. (JP application 94-167484 (1994)) has suggested hydrogels obtained, for instance, by ammonium persulphate initiated polymerisation of N-vinyl acetamide or N-vinyl formamide together with a cross-linker, such as N,N′-metylenebisacrylamide, in gel electrophoresis.
Akashi M et al. (Journal of Polymer Science: Part A: Polymer Chemistry, 31, (1993) 1153–1160) have described hydrogels based on N-vinyl acetamide.
Hashimoto et al. (JP application 95-209781 (1995) have described liquid adsorbents and tackifiers prepared by UV initiated polymerisation of N-vinyl formamide or N-vinyl acetamide together with, for instance, N,N′-butylenebis(N-vinyl acetamide) as cross-linker.
To our knowledge the problem with minimising the residual monomer amount after polymerisation is not discussed in any of these publications. Nor UV initiated polymerisation for the preparation of separation gels as defined above seems to have been described previously.